Anti-xylan sulfate polyclonal antiserum and use thereof in elisa methods

Abstract

The object of the present invention is an anti-xylan sulfate antiserum having an antibody titre of between 1/10000 and 1/15000, and obtainable using a specific immunization method, and use thereof in one or more ELISA methods for determining xylan sulfate levels in biological fluids.

1 . Anti-xylan sulfate polyclonal antiserum having an antibody titre of between 1/10000 and 1/15000. 
     
     
         2 . Anti-xylan sulfate polyclonal antiserum according to  claim 1  obtainable by the following steps:
 a) performing one or more subcutaneous injection of xylan sulfate antigen complexed with methyl-BSA and emulsified with complete or incomplete Freund's adjuvant into a mammalian; and 
 b) collecting of said polyclonal antiserum. 
 
     
     
         3 . Polyclonal antiserum according to  claim 1 , wherein said polyclonal antiserum does not recognize unfractionated heparin (UFH), low molecular weight heparin (LMW), xylan, dextran sulfate, and glycosaminoglycans selected from heparan sulfate and chondroitin sulfate A, B, C, D, or E. 
     
     
         4 . Polyclonal antiserum according to  claim 2 , wherein a) provides for 0.5-1.5 mg of antigen/injection. 
     
     
         5 . Polyclonal antiserum according to  claim 2 , wherein said mammalian is a rabbit. 
     
     
         6 . Polyclonal antiserum according to  claim 2 , wherein in step a), 5-10 subsequent injections, for a maximum period of 100-110 days are performed. 
     
     
         7 . Polyclonal antiserum according to  claim 2 , wherein in step a), injections subsequent to the first one are performed at intervals of 4-40 days. 
     
     
         8 . Polyclonal antiserum according to  claim 2 , wherein in step b), the polyclonal antiserum is collected from the mammalian after at least 100-110 days. 
     
     
         9 . ELISA method for identifying and/or quantifying xylan sulfate levels in biological fluids with the polyclonal antiserum according to  claim 1 . 
     
     
         10 . Direct or non-competitive ELISA method for the identification and/or quantification of xylan sulfate in biological fluids, characterized by the following steps:
 a. immobilization of the xylan sulfate present in the biological sample, or in titrated standard solutions, on a pre-treated solid support, through incubation for 12-20 hours at room temperature;   b. blocking the aspecific binding sites by incubation with PBS containing 10% FBS for 60 minutes at room temperature;   c. incubating with polyclonal antiserum according to  claim 1 ;   d. incubating with a rabbit IgG-specific antibody conjugated to an enzyme for 90 minutes at room temperature;   e. incubating with a solution containing the enzymatic substrate for 5-10 minutes at room temperature;   f. blocking the enzymatic reaction by addition of a denaturing solution;   g. measuring the optical density of the solution; and   h. calculating xylan sulfate concentration in the biological samples.   
     
     
         11 . Competitive ELISA method for the identification and/or quantification of xylan sulfate levels in biological fluids, characterized by the following steps:
 a. immobilizing of xylan sulfate on a pre-treated solid support, through incubation for 12-20 hours at room temperature;   b. blocking the aspecific binding sites by incubating with PBS+10% FBS for 60 minutes at room temperature;   c. incubating with both the polyclonal antiserum according to  claim 1 , and with the biological sample or with titrated solutions of xylan sulfate, for 90 minutes at room temperature;   d. incubating with a rabbit IgG-specific antibody conjugated to an enzyme for 90 minutes at room temperature;   e. incubating incubation with a solution containing the enzymatic substrate for 5-10 minutes at room temperature;   f. blocking the enzymatic reaction by addition of a denaturing solution;   g. measuring the optical density of the solution; and   h. calculating xylan sulfate concentration in the biological samples.   
     
     
         12 . ELISA method according to  claim 11 , characterized in that the color intensity developed in step g) is inversely proportional to xylan sulfate amount. 
     
     
         13 . ELISA method according to  claim 11 , characterized in that the polyclonal antiserum used in step c) is used at fixed concentration. 
     
     
         14 . The polyclonal antiserum according to  claim 2 , wherein step a) provides for 1 mg of antigen/injection. 
     
     
         15 . Polyclonal antiserum according to  claim 2 , wherein in step a), 5-10 subsequent injections, for a maximum period of 104 days are performed. 
     
     
         16 . Polyclonal antiserum according to  claim 2 , wherein in step a), injections subsequent to the first one are performed at intervals of from 7 to 14 days. 
     
     
         17 . Polyclonal antiserum according to  claim 2 , wherein in step b), the polyclonal antiserum is collected from the mammalian after 104 days. 
     
     
         18 . The method according to  claim 10 , wherein said denaturing solution is 2N sulfuric acid. 
     
     
         19 . The method according  claim 11 , wherein said denaturing solution is 2N sulfuric acid. 
     
     
         20 . The method according to  claim 13 , wherein the polyclonal antiserum used in step c) is at a dilution 1:10000.